The purpose of this research project is threefold. First, to identify and characterize the specific receptor for low density lipoprotein (LDL) on the plasma membrane of normal cells. Second, to compare the structure of normal LDL receptors to the structure of defective receptors from individuals with familial hypercholesterolemia (FH). Third, to correlate the alterations in the functions of these receptors isolated from several types of FH mutants currently available. One major approach to this problem which we are taking is to compare the protein profiles of normal and FH fibroblasts using two-dimensional polyacrylamide gel electrophoresis. Two-dimensional polyacrylamide gels are capable of resolving as many as 1000 proteins on a single gel. In addition, we have developed a new technique for double label autoradiography on polyacrylamide gels. Thus, we are able to compare, on a single gel, as many as 1000 proteins from normal fibroblasts and 1000 proteins from familial hypercholesterolemia fibroblasts. The technique is sensitive enough to detect a change as small as an alteration in a single amino acid of one of these proteins. Once a protein whose primary structure has been altered is identified, we will isolate that protein from both normal and FH cells and further analyze their structures by using two-dimensional tryptic peptide mapping. As a second approach to this problem we are characterizing the LDL receptor on the surface of normal human fibroblasts. We have been studying the interaction of a variety of lectins with the surface of fibroblast cells and the effect that the membrane bound lectins exert on the subsequent binding of LDL to the cells. We have identified several lectins which interfere with LDL binding. We will try to utilize these different lectins to prepare affinity columns in order to isolate the LDL receptor from the membranes of normal cells.